Many bacteria modulate their behavior in response to cell-cell communication in a process termed quorum sensing (Bassler, 2002). Intercellular communication is accomplished through the production, release, and detection of small signaling molecules called autoinducers. Typically, Gram-negative bacteria use acylated homoserine lactones as autoinducers, whereas Gram-positive bacteria use modified oligopeptides. In its simplest form, quorum sensing consists of the accumulation of high autoinducer concentrations at high bacterial population densities. The bacteria respond with a population-wide alteration of gene expression, allowing the community to coordinate behavior in a manner akin to cells in a multicellular organism. Quorum sensing provides a mechanism for the collective regulation of processes including biofilm formation and virulence in Pseudomonas aeruginosa and Vibrio cholerae, antibiotic production in Photorhabdus luminescences, and light production in Vibrio harveyi (Miller et al., 2001). In general, different bacterial species produce and respond to chemically distinct autoinducers, restricting quorum sensing to intraspecies communication.
Genetic and biochemical studies of quorum sensing in the marine bacterium V. harveyi led to the identification of a novel autoinducer used to control bioluminescence (Bassler et al., 1994, 1997; Chen et al., 2002; Schauder et al., 2001; Surette et al., 1999). This autoinducer signal, termed AI-2, is unusual in that it is produced by a large number of bacterial species in addition to V. harveyi. Furthermore, AI-2-responsive genes have been identified in a variety of bacteria (Xavier et al., 2003). Consequently, AI-2 has been proposed to serve as a “universal” quorum-sensing signal that enables interspecies communication (Schauder et al., 2001).
The enzyme LuxS, which has been identified in more than 55 Gram-negative and Gram-positive bacterial species, is responsible for AI-2 biosynthesis (Surette et al., 1999; Xavier et al., 2003). AI-2 signals are derived from S-adenosylmethionine (SAM), whose consumption as a methyl donor yields S-adenosylhomocysteine (SAH) (FIG. 1A). SAH is metabolized to adenine and S-ribosylhomocysteine (SRH) (Cornell et al., 1998). SRH is the substrate for LuxS (Lewis et al., 2001; Schauder et al., 2001), which cleaves it to generate homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD, FIG. 1A).
The products of the LuxS reaction strongly stimulate light production in V. harveyi (Meijler et al., 2004; Schauder et al., 2001; Zhao et al., 2003). One of these products, homocysteine, has no autoinducer activity. The other product, DPD, is expected to cyclize spontaneously to form two epimeric furanoses, (2R,4S)- and (2S,4S)-2,4-dihydroxy-2-methyldihydrofuran-3-one (R- and S-DHMF, respectively; FIG. 1B). Hydration of R- and S-DHMF would give rise to (2R,4S)- and (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R- and S-THMF, respectively; FIG. 1B).
Because DPD exists in equilibrium with other chemical species in solution (this work and Meijler et al., 2004), identifying the form that is active in AI-2 signaling in V. harveyi proved difficult. Trapping V. harveyi AI-2 in its receptor LuxP greatly facilitated its identification. X-ray crystallography allowed direct visualization at 1.5 Å resolution of the ligand bound to LuxP (Chen et al., 2002), establishing that the signal molecule is S-THF-borate (FIG. 1B). Formation of this molecule from DPD can be explained by a simple mechanism. Since borate reacts readily with adjacent hydroxyl groups on furanosyl rings (Loomis et al., 1992), it is chemically reasonable that S-THMF-borate forms spontaneously by addition of borate, which is abundant (ca. 0.4 mM) in marine environments, to S-THMF (FIG. 1B). Consistent with this scheme, chemically synthesized DPD induces bioluminescence in V. harveyi, but only in the presence of boric acid (Meijler et al., 2004). S-THMF-borate is unrelated to previously characterized autoinducers and is highly unusual in containing boron, an element rarely observed in biological molecules.
The presence of boron in the LuxP ligand raised the question of whether S-THMF-borate is the sole bacterial signaling molecule derived from DPD. Hence, this question was addressed by determining whether other bacteria that respond to AI-2 signals recognize S-THMF-borate or whether, instead, they recognize different derivatives of DPD. In the latter case, the use of S-THMF-borate as a signaling molecule might be confined, for example, to bacteria such as marine vibrios that live in relatively high-borate environments. The identification of LsrB as an AI-2 binding protein in S. typhimurium and Escherichia coli (Taga et al., 2001, 2003) provided a starting point for characterizing the spectrum of AI-2 signal molecules.
S. typhimurium carries the LuxS enzyme and synthesizes DPD. Genetic analysis has identified a set of lsr (LuxS-regulated) genes whose expression is controlled by the LuxS-generated AI-2 signaling molecule (Taga et al., 2001). The Lsr proteins appear to function in the binding, internalization, and metabolism of the AI-2 signal (Taga et al., 2001, 2003). LsrB, as suggested by its homology to periplasmic sugar binding proteins, binds the AI-2 signal directly. Other genes in the lsr operon encode LsrA, LsrC, and LsrD. These proteins form an ABC transporter complex, homologous to the ribose transporter, that internalizes the signal molecule. Internalized AI-2 is subsequently processed by additional lsr operon encoded enzymes (Taga et al., 2003). Thus, one consequence of activating the lsr operon at high cell density is that S. typhimurium clears AI-2 signaling activity from its environment. This might represent a strategy for terminating AI-2 signaling or for interfering with AI-2 signaling by other species (Taga and Bassler, 2003).
The structure of LsrB, both unliganded and in complex with its DPD-derived ligand was determined. Like other periplasmic binding proteins, LsrB undergoes a significant conformational change upon ligand binding. Most strikingly, the LsrB ligand differs from the LuxP ligand and lacks boron. Thus, two different bacterial AI-2 receptors bind chemically distinct derivatives of DPD. These findings mean that the earlier use of the term “AI-2” to refer exclusively to S-THMF-borate is not accurate. Instead, the AI-2 response in different bacterial species can be triggered by at least two different derivatives of the LuxS product, DPD.